PhcS represses gratuitous expression of phenol-metabolizing enzymes in Comamonas testosteroni R5

Abstract

We identified an open reading frame, designated phcS, downstream of the transcriptional activator gene (phcR) for the expression of multicomponent phenol hydroxylase (mPH) in Comamonas testosteroni R5. The deduced product of phcS was homologous to AphS of C. testosteroni TA441, which belongs to the GntR family of transcriptional regulators. The transformation of Pseudomonas aeruginosa PAO1c (phenol negative, catechol positive) with pROR502 containing phcR and the mPH genes conferred the ability to grow on phenol, while transformation with pROR504 containing phcS, phcR, and mPH genes did not confer this ability. The disruption of phcS in strain R5 had no effect on its phenol-oxygenating activity in a chemostat culture with phenol. The phenol-oxygenating activity was not expressed in strain R5 grown in a chemostat with acetate. In contrast, the phenol-oxygenating activity in the strain with a knockout phcS gene when grown in a chemostat with acetate as the limiting growth factor was 66% of that obtained in phenol-grown cells of the strain with a knockout in the phcS gene. The disruption of phcS and/or phcR and the complementation in trans of these defects confirm that PhcS is a trans-acting repressor and that the unfavorable expression of mPH in the phcS knockout cells grown on acetate requires PhcR. These results show that the PhcS protein repressed the gratuitous expression of phenol-metabolizing enzymes in the absence of the genuine substrate and that strain R5 acted by an unknown mechanism in which the PhcS-mediated repression was overcome in the presence of the pathway substrate.

FIG. 1

Genetic organization of the regulatory and structural genes for mPH in C. testosteroni R5. phcS (in black) was identified in this study. The other genes were identified in our previous study (50). The arrows indicate the direction of transcription. The ability (+) or inability (−) of plasmids pROR502 and pROR504 to allow P. putida PAO1c to grow on phenol as the sole carbon source is indicated. pKT231S is a derivative of pKT231 and carries a MunI-EcoRV fragment which contains the phcS gene, while pRC50Ps is a derivative of pRC50 and carries an Sse8387I-NheI fragment which contains phcR. pRC50Pk is also a derivative of pRC50 and carries an EcoRV-BglII fragment which contains a phcK promoter region. The two small arrows indicate the direction of lacZ on plasmids pRC50Ps and pRC50Pk. pSK02S carries the phcS gene which was disrupted by the insertion of a tetracycline resistance gene (Tc^r) cassette, while pBS02RS carries the phcS and phcR genes which were disrupted by the insertion of a Tc^r cassette.

FIG. 2

Effect of disrupting phcS on expression of mPH in C. testosteroni strain R5 grown on phenol or acetate in a continuous culture. (A) Phenol-oxygenating activity of strains R5 and R5S. (B) C23DOase activity of strains R5 and R5S. Each value is the mean ± standard error from two or three independent cultures.

FIG. 3

mPH expression of C. testosteroni strain R5S in response to the presence of phenol or acetate. Stationary-phase cultures grown on LB medium were exposed to MP medium with 2 mM phenol (circles) or acetate (triangles) or without growth substrate (squares). Each value is the mean ± standard error from three independent experiments.

FIG. 4

Transcriptional activity of the phcK promoter in C. testosteroni strain R5S in response to the presence of phenol or acetate. The strain was transformed with pRC50Pk, which carries a transcriptional fusion of phcKL::lacZ (white symbols) or pRC50 (control vector [black symbols]). Stationary-phase cultures were transferred to MP medium with 2 mM phenol (circles) or acetate (triangles) or without growth substrate (squares). Each value is the mean ± standard error from two or three independent experiments.